Tesi sul tema "Physiology"

Revised 06/2015. Originally published 07/2001.
3 pp.
This bulletin deals with the physiology of cotton defoliation and attempts to describe what conditions must exist inside the plant in order for defoliation to occur. It is important to understand the basic physiological processes involved in order for best crop management practices to accomplish a successful defoliation. The objectives of defoliating a cotton crop can be simply stated as: 1) to remove leaves to facilitate mechanical picking, 2) to maintain the quality of the lint, and 3) to complete defoliation with a single application of chemicals.

The practice of competitive breath-hold (apnea) diving has provided a gateway for studying the physiologic limits of severe hypoxemia and hypercapnia beyond otherwise possible in healthy humans. In elite apnea competitors, the broad objectives of this Thesis were to, a) quantify the impact of peripheral and central chemoreception, and lung volume on the elite dry-land apnea breakpoint, and b) examine the consequences of prolonged apnea on the cerebral metabolic functioning. These objectives were achieved in four experimental studies. Study 1 explored the impact of peripheral chemoreflex silencing with low-dose dopamine. Here, compared to placebo, dopamine blunted the ventilatory response to hypercapnic-hypoxia by ~27%; however, maximal apnea duration was only increased by ~5%. At the breakpoint, arterial hypoxemia was identical with dopamine compared to placebo, indicating that the apnea termination may largely be determined by a threshold level of hypoxemia to maintain consciousness. To eliminate the influence of hypoxia, Study 2 assessed the main determinants of an apnea breakpoint following hyperoxic pre-breathing. Here, the apnea duration was related to the individual forced vital capacity, and unrelated to the central chemoreflex. Respiratory muscle fatigue and pending atelectasis likely determined the capacity of a maximal hyperoxic apnea. Study 3 quantified the cerebral metabolism during apnea. The cerebral metabolic rate of oxygen, measured from the product of cerebral blood flow and the radial artery-jugular venous oxygen content difference, was reduced by ~29% at the termination of apnea. However, there was no change in the cerebral non-oxidative metabolism, calculated from the ratio of oxygen and carbohydrate metabolism. Study 4 examined the cerebral metabolic response in three apneas eliciting separate levels of hypoxemia and hypercapnia. Apneas generating the most severe hypercapnia, irrespective of hypoxia, elicited the largest reduction in the cerebral metabolic rate of oxygen. Moreover, apneas generating the most severe hypoxia, irrespective of hypercapnia, caused a cerebral net release of lactate, suggesting astrocyte glycogenolysis. Together, the findings of this thesis provide new insight into the determinants of an extreme apnea breaking point, and the observations of hypercapnic-induced reduction in oxidative cerebral metabolism provides a tenable mechanism for cerebral protection against prolonged apnea.
Graduate Studies, College of (Okanagan)
Graduate

Introduction: Varying methods of cough quantification have been used to describe cough frequency, although there is no consensus which method best relates to subjective rating of cough. With increased availability of semi-automated cough recording devices, the capability to carryout anti-tussive studies is greatly increased. However, there is no data available describing the magnitude of change in cough frequency necessary to provide therapeutic benefit. Female patients are over represented in specialist cough clinics. There is little data describing pulmonary function and airway inflammation in phenotyped patients and how these measures may relate to objectively measured cough. Airway inflammation isa common feature of chronic cough, regardless of the trigger for cough. The repetitive mechanical insult of the act of coughing may be responsible for the presence ofthis inflammation. Methods: 100 patients with unexplained chronic cough under went full diagnostic testing in order to determine potential triggers for cough. Procedures included pulmonary function testing; (spirometry, eND, EBC pH, BHR, Cough challenge, induced sputum, objective and subjective cough monitoring), Bronchoscopy, 24 hour impedance monitoring with simultaneous cough monitoring, Gastroscopy and ENT. A subset of cough recordings were quantified in cough sounds, cough seconds and cough epochs to determine the best way to quantify cough. 20 healthy volunteers performed voluntary coughing manoeuvres to determine the acute effects of coughing on airway function and inflammation. Results: Cough sounds and seconds correlate moderately with subjective and QDL . methods. Patients reported a reduced cough frequency whilst undergoing impedance testing; a reduction of33% was seen although patients were unable to quantify the scale of change. Female patients coughed substantially more than male patients, with the largest difference seen at night. Cough frequency was predicted by gender, C5 and age. Cough frequency was not related to a specific trigger for cough. Reduced small airway flows were seen that were independent of BHR. Prominent airway neutrophilia was seen regardless of the trigger for cough. Acute changes in eND and EBC were seen after short periods ofvoluntary coughing. Sputum inflammatory mediator levels showed huge variability and did not change as a result of coughing. Bronchodilation ofthe small airways was also seen after coughing. Conclusion: The use of objective cough monitoring has enabled me for the first time to describe the most appropriate current methods of cough quantification and to demonstrate the magnitude ofreduction in cough frequency that is appreciated by -patients.-I-havereportedimportant significant genderdifferences in cough frequency and shown that cough frequency is predicted not only by gender but also age and cough reflex sensitivity showing important implications for underlying mechanisms of chronic cough. I have also shown for the first time that pulmonary function measures are reduced in patients without indication of asthma. The mechanical act of coughing causes acute bronchodilation of small airways and reduces eND and EBC pH.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2016.
Cataloged from PDF version of thesis.
Includes bibliographical references (pages 125-133).
Using molecular imaging in vivo, biomolecular and cellular phenomena can be investigated within their relevant physiological context, addressing a central challenge for 21st century biomedicine and basic research. To advance neuroscience in particular, molecular-level measurements across the brain inside the intact organism are required. However, existing imaging strategies and available probes have been limited by serious constraints. Magnetic resonance imaging (MRI) provides deeper tissue penetration depth than optical imaging and better spatial resolution and greater versatility in sensor design than radioactive probes. The most important drawback for MRI probes has been the need for high concentrations in the micromolar to millimolar range, leading to analyte sequestration, complications for noninvasive brain delivery, and toxicity. Efforts to address the sensitivity problem, such as nuclear hyperpolarization, introduce their own technical constraints and so far lack generality. Here, we introduce a conceptually novel molecular imaging technique based on artificially induced physiological perturbations, enabling molecular MRI with nanomolar sensitivity. In this imaging strategy, we take advantage of blood as an abundant endogenous source of contrast compatible with multiple imaging modalities including MRI and optical imaging to decouple the concentration requirement for molecular sensing from the concentration requirement for imaging contrast. Highly potent vasoactive peptides are engineered to respond to specific biomolecular phenomena of interest at nanomolar concentrations by inducing dilation of the microvasculature, increased local bloodflow, and consequently, large changes in T₂*-weighted MRI contrast. This principle is exploited to design activatable probes for protease activity based on the calcitonin gene-related peptide (CGRP) and validate them for brain imaging in live rats; to use CGRP as a genetic reporter for cell tracking; and to create fusions of a vasoactive peptide from flies to previously characterized antibodies capable of crossing the blood-brain barrier (BBB), suggesting the possibility of minimally invasive brain delivery of such probes. We demonstrate the feasibility of highly sensitive molecular MRI with vasoactive probes at concentrations compatible with in situ expression of probes and delivery across the BBB, and show that vasoactive peptides are a versatile platform for MRI probe design which promises unprecedented in vivo molecular insights for biomedicine and neuroscience.
by Adrian L. Slusarczyk.
Ph. D.

The aim of this thesis is to bring together and summarize the results of twenty-five years of active research into the immunology and physiology of the mammalian lymphoid system using the sheep as the model species. For this work I have exploited the cannulation of peripheral lymphatics, which enables the monitoring of lymph, lymphocytes and dendritic cells that are constantly trafficking from the skin or from lymph nodes. The use of this technique in the sheep permits access to large numbers of lymph-borne cells over extensive periods and in a form far closer to their in vivo non-activated state than from any other species. I have organized the publications into four distinct, but interrelated chapters. Chapter 1 is concerned with the physiology of sheep lymphoid cells and describes the use of the cannulated lymphatic model to answer fundamental questions of lymphoid biology. My earliest work was focused on the non-random migration of lymphocytes and the identification of two lymphocyte populations; one associated with the gastrointestinal tract and other mucosal organs and the other with peripheral lymph nodes and the spleen. Later work identified two separate populations of B cells with distinct recirculation properties and also concentrated on the lymph node response to antigen and the role played by antigen in modulating lymphocyte recirculation. Much of my work in the last few years has been concerned with the biology of dendritic cells (DCs), the cell population uniquely able to induce the primary immune response. The "pseudo-afferent" cannulation system in sheep is, arguably the best system for this study, as the isolation procedure does not lead to aberrant changes in cell phenotype and function. Chapter 2 relates the work to characterize the sheep immune system, in order to exploit further the sheep as a species for immunological study. Much of my efforts involved the production and characterization of anti-sheep MHC and CD1 monoclonal antibodies (mAbs). This resulted in the generation of monoclonal reagents that are now the standards used to define the ovine/bovine homologues of MHC class I, and class II and CD1.

Kyoto University (京都大学)
0048
新制・課程博士
博士(人間・環境学)
甲第13146号
人博第353号
新制||人||88(附属図書館)
18||D||154(吉田南総合図書館)
UT51-2007-H419
京都大学大学院人間・環境学研究科共生人間学
(主査)教授 森谷 敏夫, 教授 小田 伸午, 助教授 林 達也
学位規則第4条第1項該当

Background: Cardiovascular disease is often characterised by functional and structural changes in the blood vessel wall, usually associated with endothelial dysfunction. Therefore, interest in targeting the dysfunctional endothelial cell has heightened. Ca2+ signalling is crucial to endothelial cell physiology as it drives a number of intracellular signalling pathways. Store-operated Ca2+ entry (SOCE) mediated by Orai1 channels is of particular interest as it provides a major Ca2+ influx pathway in endothelial cells, driving cell migration and proliferation, ultimately contributing to endothelial repair and integrity, angiogenesis and wound healing events. The novel hypothesis is that modulation of Orai1 channels will have important physiological effects on endothelial cell function. Methods and Results: A novel series of Orai1 inhibitors has been identified in this thesis with improved pharmacological and physicochemical properties compared to existing SOCE inhibitors. Optimisation of compounds’ structure-activity relationships (SAR) via Ca2+ measurement assays using human umbilical vein endothelial cells (HUVECs) has revealed important insights into functionally important features of SOCE blockers. In conjunction with in silico modelling, a novel binding site in the Orai1 channel located in a small extracellular pocket has been proposed. The generation and subsequent testing of a quaternary ammonium analogue of the parent compound, JPIII supports the hypothesis. The novel SOCE inhibitors suppressed endothelial cell migration and proliferation without affecting cell viability. Reassuringly, the small molecules were well tolerated in vivo, supporting further development and testing of such blockers in animal models of cardiovascular disease. Here, a novel transgenic murine model with the potential for temporal and conditional disruption of Ca2+ permeation in Orai1 channels has been generated and characterised, offering new possibilities for better understanding of the physiological and pathological roles of Orai1 and the therapeutic potential of targeting this channel. Conclusion: Novel SOCE inhibitors have been characterised in vitro, their mechanism of action interrogated and their properties optimised for the next stages of in vivo testing in an animal model of cardiovascular disease.

Water is a critical resource for which plants compete in many terrestrial communities. In arid communities where water most limits plant growth, rainfall events occur in discrete, pulsed events. These pulses of water create highly variable soil moisture availabilities. Plant species respond differently to variation in soil water availabilities throughout a season and between years. How species vary in their responses to a range of water availabilities is thought to influence community and ecosystem properties. Many previously proposed hypotheses are not suitable to explain rapidly fluctuating resource availabilities or numerous input events throughout the growing season. This dissertation uses variation in water availability as a model resource to examine how species characteristics influence the process of exploitation competition within plant communities.Experiments were conducted to examine variation in growing season, exploitation competition between several pairs of co-occurring species in the Sonoran desert. Three separate studies evaluated several components of community dynamics thought to be influenced by exploitation competition. Spatial attributes of exploitation competition were assessed by measuring the performance of a deep-rooted species across the boundary of a natural expansion of a shallowly rooted species. Then, neighborhood composition was varied for species of similar growth-form to address the affects of species characteristics to shifts in abundances under field conditions. Lastly, species from the neighborhood composition study were placed under controlled, manipulated water availabilities to measure their fundamental operational conditions.Performances of plant species in all experiments were assessed using a combination of physiological and vegetative measurements, capturing the responses of the plants to both the dynamic growth conditions during the growing season, and integrated measures of plant performance post growth season. A shared preference was found for all species, where the performance of all species was greatest when water was most available in the soil profile. This work suggests the mechanism within a functional type by which plants coexist at various abundances is in part due to the variation in responses to temporal resource gradients. The variation in availability of resources and the species composition within the community should be considered in studies of competition between plant species.

Whilst mechanical impact is known to be essential for cartilage maintenance, it has been noted that altered joint loading and increased force may lead to cartilage degradation and increase the risk for the development of osteoarthritis (OA). This study investigated the cellular responses of chondrocytes to mechanical impact, and the effects of possible chondroprotective agents for OA preventative strategies in individuals exposed to high impact, repetitive loading. Single-impact mechanical trauma (force 1.14 N, pressure 6.47 KPa) was determined to induce biphasic decrease in cell volume to 647.38±60.38 μm3 at 2 h and 516.52±38.86 μm3 at 48 h, the initial phase of which was observed to be an active mechanotransduction mechanism, termed Impact-Induced Volume Decrease (IIVD), and the subsequent phase to be Apoptotic Volume Decrease (AVD). The newly defined IIVD was concluded to be dependent upon the PKC/PLCβ3 pathway, and possibly mediated by intracellular Ca2+ store release and Volume Sensitive Organic Anion Channel (VSOAC) activity. Furthermore, mechanical impact was observed to induce a rapid decrease in F-actin from 1.19±0.13 MU to 0.87±0.02 MU, termed Impact-Induced Actin Decrease (IIAD) and associated with the biphasic rise in cell death at rates of 2.75±0.41 %.h-1 and 0.66±0.03 %.h-1. Both in vivo exercise and in vitro mechanical load induced a release IL-1β (20.67±2.58 % and 5.86±0.21 AU), MCP-1 (25.69±0.53 % and 1.45±0.01 AU) and IL-10 (8.97±2.40 % and 5.55±0.28 AU), with in vivo concentrations correlating with joint magnitude and strike patterns. Decreased levels of IL-1β and MCP-1 (to 9.60±2.34 % and 9.01±2.34 %, respectively) observed in the evening were further confirmed using a hyperosmotic-treated in vitro model of prolonged static-loaded cartilage with evidence for a IL-1β-dominated paracrine loop between articular cartilage and mononuclear phagocytes. In vitro, chondroprotective and antiinflammatory actions of chondroitin sulphate, glucosamine sulphate, REV 5901 and Tamoxifen were associated with a reduction in pre-impact cell volume (average of 31.91±4.19 %) and increased pre-impact actin levels (average of 39.92±9.29 %). Anti-inflammatory agents, curcumin and dexamethasone exhibited less effective chondroprotective actions, via inhibition of IL-1β (average of 83.45±1.30 %) and thus apoptosis. To conclude, high impact exercise is recommended with a place for chondroprotective properties of chondroitin, glucosamine sulphate and/or curcumin in high-risk groups before OA onset.

Der Stellenwert feinmotorischen Könnens und Handelns steigt im Zeitalter der globalen Vernetzung und Technisierung kontinuierlich an. Das Erlernen solcher motorischer Abläufe unterliegt vielen Einflüssen und Störgrößen. Aus vorangegangenen Arbeiten ist bekannt, dass die Atmung nicht nur mechanische, sondern auch zentralnervöse Einflüsse auf die Geschwindigkeit und Genauigkeit anderer Muskelaktivitäten hat. In der vorliegenden Arbeit wurden an 16 Probanden mittels sprungförmiger Fingerfolgetests Lerneffekte bei feinmotorischen Bewegungen in Abhängigkeit von der Atmungsphase untersucht. Die Dauer eines Versuches lag im Mittel bei 15 Minuten mit einer Sprunghäufigkeit von ca. 8 Sprüngen pro Minute. Ziel war es, einer digital über Monitor präsentierten sprungförmigen Vorgabefunktion durch eine Fingerbeugung so schnell und so genau wie möglich zu folgen. Die Folgegenauigkeit – dargestellt durch den Regelfehler – verbesserte sich im Laufe des Versuchs nur geringfügig (p = 0.6). Die Kennwerte der Geschwindigkeit (Latenzzeit, Anstiegszeit) wurden dagegen über die Versuchszeit schlechter. Letzterer Befund lässt sich eher als Folge von Ermüdung und nachlassender Konzentration interpretieren. Unter Berücksichtigung der Atmungsphase konnten wir dagegen einen positiven Lerneffekt mit signifikanter Verbesserung der Folgegenauigkeit bei den Sprüngen nachweisen, die in der frühen Exspiration ausgelöst wurden. Dieses Ergebnis zeigt, dass feinmotorisches Lernen atmungsphasenabhängig moduliert wird.

Laboratory experiments on the physiological response of members of the nanophytoplankton to temperature and light limitation and nutrient saturation were conducted in order to investigate if nanophytoplankton conforms to Plankton Functional Types (PFTs) for modelling purposes. This thesis concluded that nanophytoplankton does not follow all of the assumed physiological traits. The Q10 estimates for members of the nanophytoplankton are considerably lower than Eppley, and since nanophytoplankton does not follow the Eppley curve at warmer temperatures, the results suggest that the Eppley assumptions cannot be used to describe nanophytoplankton. μmax0 is used as a temperature physiological modelling parameter (as well as Q10) which are components of the exponential and linear fits. However, nanophytoplankton best fits to an optimum function which uses μopt, Topt and dT as model parameters. These results are in contrast to the Eppley assumptions. Using a dynamic photosynthesis model five phytophysiological parameters were derived including the maximum photosynthesis rate (Pcm,), respiration rate (resp), the initial slope of the line (achl), light inhibition (βchl) and the maximum chlorophyll to carbon ratio (θmax). These parameters were estimated using an acclimated model which used the instantaneous rates of photosynthesis to estimate the other parameters. The acclimated model gave the best fit (AIC = -3.75 vs. = -0.95). These results are in contrast to those used for PFT modelling purposes. Parameters are comparable for Pcm, resp and θmax but showed significant differences for αchl and βchl the latter of which was underrepresented in the dynamic model, and the former of which is used as a model parameter for PFT parameterization. Chlorophytes had stronger light inhibition (mean βchl= 0.72 g C m2 (mol photons g Chl α)-1) than haptophytes (mean βchl = 0.34 g C m2 (mol photons g Chl α)-1). βchl is significantly lower for haptophytes (P = 0.002). Members of the nanophytoplankton showed relatively high μmax (0.81 d-1 from the acclimated model fit) and mean photosynthesis rates 1.8 Pcm (d-1) mean cell volume 37 μm3). Maximum growth rates increased with increasing cell volume for all of the species. Members of the nanophytoplankton alter their elemental stoichiometry and assimilated nutrients in excess of their requirements but as a PFT, there were no statistically significant deviations from Redfield. Under nutrient replete conditions Chl α:C increased linearly with increasing temperature and increased linearly with decreasing light. Overall, these results suggest that further physiological data is required in order to parameterize models to estimate nanophytoplankton physiological responses to climate change.

The cold season in the Arctic extends over eight to nine months during which ecosystem gas exchange and water balance of arctic plants have been largely unexplored. The overall objective of this thesis was to examine two critical gaps in our knowledge about tundra cold season processes – ecosystem respiration at very low temperatures and water uptake during the winter-spring transition. I determined the temperature response of ecosystem respiration of tundra monoliths down to temperatures as low as can be expected under snow-covered conditions (-15 °C). Temperature responses fit the Arrhenius function well with Q10 values over the range of -15 to 15 °C varying from 6.1 to 4.8. I used deuterium-enriched water (2H2O) as a tracer to evaluate water uptake of evergreen plants at snowmelt when soils are largely frozen. The results revealed that evergreen plants take up water under snow cover, possibly via roots but undoubtedly by foliar uptake.

Background Highest rates of exertion related cardiac death occur during cold air inhalation (CAI): e.g. shovelling snow, but the pathophysiology is unclear. Coronary micro-vascular resistance (MVR) is the major factor regulating coronary blood flow and subsequent myocardial perfusion. Patients with significant coronary artery disease may be more prone to adverse events due to a reduced vasodilator reserve. Novel intracoronary wires, that simultaneously measure coronary artery pressure (Pd) and coronary blood flow (CBF) allow quantification of MVR, enabling physiological investigation of the effects of CAI during exercise. Study 1. hMR versus IMR at predicting microvascular dysfunction. We compared headto- head, the diagnostic accuracy of the two available invasive indices of MVR, Dopplerderived hyperaemic microvascular resistance (hMR) versus thermo-dilution-derived index of microcirculatory resistance (IMR), at predicting microvascular dysfunction. We then used the most accurate measure of MVR in Study 2. Study 2. Cold air with and without exercise on MVR in CAD patients. We explored the effects of CAI alone and during exercise on MVR and CBF in patients with significant coronary artery disease. Methods Study 1. 56 patients (61 ± 10 years) undergoing cardiac catheterisation for stable coronary artery disease or acute myocardial infarctions (AMI) were recruited. Simultaneous intracoronary pressure, Doppler flow velocity and thermodilution were carried out in 74 vessels without obstructive epicardial disease, at rest and during hyperaemia. In the absence of a gold-standard, the following three measures of microvascular dysfunction were used, using a pre-defined dichotomous threshold for each parameter: 1) Mean coronary flow reserve (CFRmean), defined as the average value of Doppler and thermodilution derived coronary flow reserve 2) Cardiac Magnetic Resonance (CMR) defined myocardial perfusion reserve index (MPRI) 3) CMR defined extensive microvascular obstruction (MVO). Study 2. 35 patients (62 ± 9 years) with significant coronary artery stenoses who were undergoing coronary angiography were allocated to 5 minutes of either: 1. CAI (-15oC), n=10 2. Exercise (Incremental supine ergometry), n=24 3. Exercise with CAI, n=13. (12 patients did both conditions 2. and 3., and for these patients the order was randomised). Forty seven datasets were obtained in total. We compared rest and peak measurements of Doppler-derived MVR (Pd/CBF) and CBF. We also used wave intensity analysis to identify waves that accelerate and decelerate CBF, and calculated the proportional contribution of accelerating waves as a coronary perfusion efficiency index. Results Study 1. hMR had better diagnostic accuracy than IMR to predict CFRmean (area under curve, (AUC) 0.82 versus 0.58, p < 0.001, sensitivity/specificity 77/77% versus 51/71%) and MPRI (AUC 0.85 versus 0.72, p=0.19, sensitivity/specificity 82/80% versus 64/75%). In AMI patients, the AUCs of hMR and IMR at predicting extensive MVO were 0.83 and 0.72 respectively (p=0.22, sensitivity/specificity 78/74% versus 44/91%). Study 2. MVR increased during CAI alone, whereas MVR decreased during exercise. Exercise with CAI was associated with less decrease in MVR. The increase in CBF was similarly less during exercise with CAI versus without. Coronary perfusion efficiency increased during exercise. However the addition of CAI during exercise abolished this. Conclusion Study 1. In our study cohort Doppler-derived hMR had superior diagnostic accuracy over IMR at predicting several invasive and non-invasive measures of microvascular function. This measure was therefore used to measure MVR in Study 2. Study 2. In CAD patients CAI substantially attenuated the reduction in MVR and the increase in CBF that normally occur during exercise. Moreover, while the heart has improved coronary perfusion efficiency during exercise, this may be attenuated when exercise is combined with CAI. This suggests that CAI during exercise may impede coronary vasodilatation and ventricular relaxation, rendering the heart more susceptible to ischaemia. Complementary studies (Studies 3 and 4) were performed in the absence of invasive measures of central and coronary haemodynamics to examine the differential effects of isometric and dynamic exercise (Study 3) and that of first and second exercise efforts (Study 4).

Thesis (D. Sc.)--Faculty of Science, University of Adelaide, 1990.
Published works [representing] original research conducted during the various phases of [his] academic development--Pref. Includes bibliographical references.

Plasma cells are central to an effective immune response, being the sole producers of the antibodies, yet they can caqse severe disease in autoimmunity and multiple myeloma. Therefore their differentiation and survival must be tightly regulated. In this work I investigated some of the mechanisms regulating plasma cell differentiation and life span. The differentiation of a long lived B cell to a short lived plasma cell entails a profound structural and functional metamorphosis finalized to the massive production of immunoglobulins (Ig). Exuberant Ig synthesis causes several types of stress in differentiating plasma cells. My work deals with the characterization of. the C/EBP transcription factor CHOP in plasma cell differentiation. Comparing differentiation of B cells harvested from chop'!' mice to wt cells I found a mild phenotype, consisting in an increased accumulation of intracellular IgM aggregates and a decreased secretion of this antibody class, in vitro and in vivo. These findings' reveal a novel role for CHOP in ensuring optimal functionality of the secretory pathway in the course of plasma cell differentiation. CHOP is involved in the differentiation of various cell types, where it interacts with other members of the C/EBP family favoring or impeding differentiation and it is an important factor in the ER stress response named unfolded protein response (UPR), in which it plays a pro-apoptotic role in most of the systems tested. I extended my investigation on the functions of CHOP in B cells by examining the resistance to ER stress-induced apoptosis in wt and in chop-I' cells. Surprisingly, I observed that in B cells CHOP expression in the UPR plays an anti-apoptotic function. Altogether my data suggest a cell-type specific role for CHOP in B cells and add information on the multi-faceted role of this transcription factor. Most plasma cells exhibit a short life span. The mechanisms at the basis of plasma cell apoptosis are still obscure. I propose that multiple forms of stress, linked to the massive antibody production, contribute to plasma cell death.

Peptidases regulate important physiological processes by controlling levels of bioactive peptides and occasionally through noncatalytic processes. This thesis presents a study of prolyl endopeptidase-like (PREPL), which is a peptidase involved in several human deletion syndromes, including hypotonia-cystinuria syndrome (HCS). Phenotypes tentatively attributed to PREPL deletion include hypotonia and decreased growth hormone (GH) levels. However, little is known about the mechanisms by which PREPL deletion causes these phenotypes. To better understand PREPL catalytic activity, we used an activity-based protein profiling fluorescence polarization screen to identify the first specific PREPL inhibitors. We proceeded to demonstrate the activity of these inhibitors in cells and discovered several classes of cell-active PREPL inhibitors. Further, one of these inhibitors, 1-isobutyl-3-oxo-3,5,6,7-tetrahydro-2H-cyclopenta[c]pyridine-4-carbonitrile, was able to enter mouse brains. To characterize PREPL substrate specificity, we performed several substrate profiling experiments, but no substrates could be identified, in line with reports from other groups who used related approaches to attempt to identify PREPL substrates. To characterize any noncatalytic functions of PREPL, we used an affinity purification-mass spectrometry approach (AP-MS) to search for any protein-protein interactions of PREPL. We identified brain-expressed X-linked 2 (BEX2) as a novel interactor of PREPL, and confirmed this interaction by immunoblot. Several other proteins identified in the AP-MS experiment, including several members of the STRIPAK complex are being further investigated for possible PREPL interaction. To determine whether HCS phenotypes are in fact due to PREPL deletion and to delineate the molecular pathways involved, we generated a conditional PREPL knockout mouse. These mice were visibly smaller than wildtypes and growth curve analysis verified that from week three of life, there was a significant difference in weight between wildtype and knockout mice. Initial surface righting task experiments also indicate that PREPL knockout pups may have a hypotonia phenotype. In summary, we have developed several new tools for studying PREPL catalytic and noncatalytic function, demonstrated that PREPL deletion causes a GH-related growth deficiency and possible hypotonia and thus moved several steps closer to understanding the molecular mechanisms underlying PREPL deletion phenotypes.
Chemistry and Chemical Biology

CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO
A pesquisa procura investigar como um estágio supervisionado na formação de professores acontece em uma escola de educação básica. Este estudo tem como base o projeto O estágio nos cursos de formação de professores como uma via de mão dupla entre universidade e escola, que buscou juntar todos os envolvidos no estágio - estagiários, professores supervisores de estágio e professores regentes - para a discussão conjunta sobre o papel do estágio na formação de futuros docentes. Os sujeitos centrais desta pesquisa são aqueles envolvidos nesse projeto, em seu primeiro semestre de desenvolvimento, ao redor de dois cursos de formação de professores da PUC-Rio, Geografia e Letras (Português-Inglês), e de uma escola municipal do Rio de Janeiro. Foram entrevistados oito estagiários, quatro professores supervisores de estágio, dois professores regentes e a diretora da escola. Durante dois meses, as aulas dos dois professores regentes, das respectivas licenciaturas, foram observadas com e sem a presença de seus estagiários. Além disso, as reuniões e encontros com todos os envolvidos foram acompanhados, na tentativa de entender como o estágio se desenvolvia em todas as suas dimensões. A literatura que entende a escola como espaço real da formação inicial e continuada e do desenvolvimento profissional de professores, como se observa em Nóvoa, Perrenoud, Canário, Roldão, Tardif, Lüdke, entre outros, dá sustentação à pesquisa, juntamente com a literatura sobre estágio de Ghedin, Pimenta e Lima. A análise do estágio já foi feita pelos trabalhos de Cardozo (2003) e Albuquerque (2007), um pela ótica dos alunos estagiários, outro pela ótica dos professores regentes. A presente dissertação analisa o estágio supervisionado pela ótica da escola, contribuindo, assim, para sua análise do estágio em três diferentes perspectivas. As constatações indicam que o estágio, no contexto aqui estudado, constitui rica possibilidade de troca entre os envolvidos, mobilizando todos, inclusive os alunos da escola, ou seja, provocando algum tipo de movimento interno de cada sujeito. O estágio observado revelou-se também como mais significativo e menos burocrático para os estudantes, favorecendo a imersão desses futuros professores em seu contexto de trabalho. Constata-se ainda que a aproximação entre todos os sujeitos, estagiários, supervisores de estágio e professores da escola, em uma proposta conjunta de estágio, permite entender o cruzamento de saberes entre universidade e escola. A adesão de todos os envolvidos no estágio a um projeto comum abre possibilidades para se pensar em futuras propostas de formação docente em real colaboração entre estas duas instituições formadoras de professores.
This research seeks to investigate how a supervised teaching practice occurs at a secondary school during teacher education. The study is based on the project Teaching practice in courses for teacher education as a two-way road between university and school, which has sought to bring together those who are involved in teaching practice, such as future teachers, teacher trainers and school teachers, in order to discuss with the group the role of teaching practice in the training of future teachers. The central subjects of this research are those who are involved in the above mentioned project, mainly in its first six months. They come from two teacher education degree courses for teachers in PUC-Rio - Geography and Languages (Portuguese-English) - and from a public sector secondary school in Rio de Janeiro. We interviewed eight future teachers, four teacher trainers, two school teachers and the principal of the school involved. During two months, we observed the classes with or without the presence of future teachers. Besides, we have participated in every meeting with the people involved in the project, in the attempt to understand how the teaching practice would develop as a whole. Our bibliographical support is the literature on school as a real space of initial and continual training and of teacher professional development, as it is possible to learn from Nóvoa, Perrenoud, Canário, Roldão, Tardif and Lüdke among others. We also based our study on the literature on teaching practice taken from Ghedin, Pimenta and Lima as well as on the analysis of teaching practice that has already been carried out by Cardozo (2003) and Albuquerque (2007), the former from the perspective of future teachers, and the latter from that of the school teachers. This dissertation analyzes teaching practice departing from the school, contributing to the analysis of supervised teaching practice from three different perspectives. The analyses indicates that teaching practice, in the context in which we have studied, constitutes a rich opportunity for interchange between those who are involved in it, mobilizing everyone, including the students attending the school; that is, leading to some kind of internal movement of every subject. The observed teaching practice also reveals itself as more significant and less bureaucratic for the trainees, contributing to the immersion of these future teachers in their job context. It is also possible to observe that the approximation between the subjects of the teaching practice, such as future teachers, teacher trainers and school teachers, allows us to understand the movement of knowledge between university and school. The engagement of everyone involved in the current teaching practice process in a common project opens possibilities for thinking about future proposals for teacher education in real collaboration between both teaching institutions.

Background: Nowadays, in paediatric surgery, intra-abdominal procedures are commonly performed with a laparoscopic approach. Nevertheless, the effects and advantages of laparoscopy in children have not been extensively investigated. Aims of this thesis are: 1) To quantify the absorption of carbon dioxide (C02) during laparoscopy; 2) To investigate if laparoscopy provides benefits compared to open surgery in the management of common surgical conditions in children. Methods: The thesis includes two parts: one part focuses on the absorption of C02 during laparoscopy. The second part focuses on the outcome of laparoscopy. Data were obtained by investigating two common surgical procedures: the Nissen fundoplication for treatment of gastro-oesophageal reflux (GOR) and the Ramstedt pyloromyotomy for treatment of pyloric stenosis. For the laparoscopic Nissen fundoplication, a follow-up study on a randomised controlled trial (RCT) including 38 children was performed. Also, a large review on patients who underwent a second operation (redo-Nissen fundoplication) for recurrent GOR was performed. For the laparoscopic pyloromyotomy, a double blind, multicentre, international, RCT was performed enrolling 180 children. Results: the work in this thesis demonstrates that 10-20% of C02 eliminated during laparoscopy in children is derived from the absorption through the peritoneum. The results of the RCT comparing laparoscopic and open Nissen fundoplication shows that this antireflux procedure improves the quality of life and controls GOR independently of the technique used. The laparoscopic technique is associated with an improvement of gastric emptying in the post-operative period and lower incidence of retching at 4-year follow-up. In children requiring redo-Nissen fundoplication, there is a high failure rate and redo-fundoplication after primary laparoscopic Nissen has lower risk of failure. The RCT comparing laparoscopic with open pyloromyotomy reveals that both procedures are successful. The laparoscopic pyloromyotomy provides a shorter post-operative recovery, lower analgesia requirement and higher parental satisfaction.

Cilia are cellular organelles that generate microfluidic flow at multiple sites in the body. They are important for health due to their critical roles in mucus clearance in the respiratory tract, circulation of cerebrospinal fluid in the ventricles of the brain, transport of ova in the Fallopian tubes, and left-right patterning of the body. Nonetheless, standards for basic mechanical phenotyping of cilia are still relatively undefined. The aim of this thesis is to develop an experimental and conceptual framework for comprehensive ciliary phenotyping. Towards that aim, we pursue three major lines of investigation involving ciliary physiology, pathophysiology, and measurement.

Our investigation into pathophysiology looks at the possibility of quantifying intermediate ciliary flow defects. Specifically, we investigate subtle changes to ciliary flow generated by genetic knockdown of ciliary proteins, alterations in chemical signaling, and changes to the viscous environment of ciliated surfaces. We additionally quantify the onset of ciliary flow in the context of normal development.

Secondly, we demonstrate the use of optical coherence tomography (OCT)-based velocimetry techniques for the measurement of cilia-driven fluid flow. In particular, we focus on a class of correlation-based techniques that utilizes the complex OCT signal to recover the total speed of fluid flow. We analyze and extend these techniques towards directional velocity measurements, and eventually towards quantification of the full three-dimensional, three vector component velocity flow field.

Finally, our investigation into ciliary physiology involves the development of a simplified model of ciliary function. Building on previous models of ciliated surfaces as shearing elements, we develop our "treamdill-in-a-pool" model of ciliated surface that also incorporates the dynamics of functional reserve and failure. These efforts motivate the measurement of three important physical parameters, flow rate, force, and mechanical power output, under conditions of increased viscous loading.

Building on these themes, we propose a comprehensive, optical-imaging based approach towards quantifying flow, force, and power in the context of ciliary performance and failure. We present a method of quantifying these mechanical properties not by direct measurement, but rather by inference from the fluid flow field that is generated by ciliary action. In all, we propose a new theoretical and experimental framework for biomechanical phenotyping of ciliated surfaces.

Physiological and morphological aspects of acclimatization were studied in cultured tomato (Lycopersicon esculentum Mill.), banana (Musa accuminata L.) and date palm (Phoenix dactyli/era ). The nutrient availability from agar solidified culture medium was determined to establish the nutrient status of the cultured plandets before transfer to ex vitro conditions. Analysis of the plant tissues demonstrated decreasing tissue concentrations of the major elements nitrogen, phosphorus and potassium with decreasing concentration of basal salts in the medium. The effects of agar and increasing sodium concentration in the culture medium was studied in cultured banana plants. Plandets grown on agar solidified medium with increased levels of sodium, exhibited reduced growth and stomatal movement. The use of agar as a solidifying agent was shown to reduce root growth, development and stomatal functioning in these plants. The efficiency of ion and water uptake, and translocation in in vitro and acclimatized tomato plants was assessed using [32P]-orthophosphate and [3H]_ tritiated water. The functional capacity of the root system fOlmed in vitro was established, and assessed following acclimatization treatments at 40% and 80% relative humidity. Comparative studies with tomato seedlings demonstrated reduced efficiency of ion translocation to the shoot in plandets growing in vitro. However, transport to the shoot improved during acclimatization. Ion absorption studies on in vitro and acclimatized palm plants demonstrated phosphate uptake and translocation in both plant types. A detailed examination of the tissue structure through the root/shoot junction and roots of · cultured, acclimatized and seedling tomato plants illustrated differences in the vascular development between the three plant types. However, no major abnormalities were observed which could have accounted for the reduced translocation efficiency in the cultured plants. Increased vascularization present in the root/shoot junction of the cultured plants may increase resistance to the transpiration flow through the region. The type of root system produced in vitro and the root/shoot ratio was manipulated using varying IAA and sucrose treatments. Improved root development and plantlet survival rates were achieved by reduced exposure to IAA during the root initiation phase followed by root elongation on IAA free medium supplemented with sucrose. Acclimatization at low relative humidity (40%) was achieved by producing plandets with balanced root/shoot ratios and a well developed root system.

This thesis is concerned with a very common disorder of iron homeostasis: iron deficiency. The specific focus is the manner in which iron deficiency influences physiological responses to hypoxia in humans. This work is predicated on observations made over many decades in vitro and in vivo, suggesting that variations in the bioavailability of iron have important consequences for certain biological processes known to depend on oxygen availability. Three separate but related studies together form the basis for this thesis. The first two, Study A and Study B, adopt a similar approach in recruiting healthy volunteers who differ according to iron status, yielding iron-deficient and iron-replete groups in both cases. In Study A, the behaviour of the pulmonary circulation is investigated during a sustained hypoxic exposure, before and after an intravenous infusion of iron. In Study B, skeletal muscle metabolism is explored, both at the level of high-energy phosphate metabolism and the integrated physiological responses to exercise on a cycle ergometer. In the third study, Study C, a different approach is taken, recruiting patients with chronic obstructive pulmonary disease (COPD), and exploring the prevalence and associations of iron deficiency in this condition. Chapters 2 and 3 describe experiments using sustained hypoxia in a normobaric chamber, during which the pulmonary circulation is assessed non-invasively using Doppler echocardiography. These reveal augmented hypoxic pulmonary vasoconstriction (HPV) in iron-deficient individuals, who also exhibit greater sensitivity to the effects of an infusion of intravenous iron. Additionally, the way in which certain circulating mediators important for iron haemostasis change over the course of these hypoxic exposures, and how iron status influences these responses, is explored. Chapter 4 reports the findings of experiments using 31P-magnetic resonance spectroscopy and cardiopulmonary exercise testing, which demonstrate abnormal whole-body metabolism in iron-deficient individuals during large muscle-mass exercise, despite the absence of a clear defect in mitochondrial oxidative phosphorylation. Intravenous iron is found to have significant effects to alter the lactate threshold in healthy individuals, but the effects are more striking in iron-deficient individuals. Collectively, these experiments imply that iron deficiency promotes a more glycolytic phenotype. Chapter 5 explores iron deficiency in COPD, a condition in which pulmonary vascular disease, hypoxia and skeletal muscle dysfunction coexist, and examines some of the difficulties in assessing iron status in the setting of a chronic inflammatory disorder. Iron deficiency is found to be common, and unexpectedly associated with significantly more severe hypoxaemia, in patients with COPD. Possible reasons for these findings, and their clinical implications, are considered. Chapter 6 provides a summary of the main conclusions to be drawn from the studies presented in this thesis.

Using a high throughput closed respirometry method to measure oxygen consumption, I determined metabolic rates in asexual and sexual Schmidtea mediterranea and Girardia dorotocephala, as a function of temperature, taxon, stressors, reproductive mode, age, regeneration, and specific dynamic action. This study has shown that oxygen consumption can reliably be measured in planaria using optode closed respirometry, and also provided a reliable method for measuring wet mass in planaria, which has been a challenge to researchers in the past. This research revealed that oxygen consumption in S. mediterranea is 1.5-2.1X greater in the sexual strain over the asexual strain at 13-18°C. Within the sexual strain, oxygen consumption is 1.5 -2.2X greater in sexually mature adults over the sexually immature groups (hatchlings, juveniles, and regenerating sexuals). Furthermore, I was able to quantify differences in sexual morphology between these groups exhibiting significant differences in oxygen consumption. The results of this research supports a theory of higher metabolic costs with sexual maturity in S. mediterranea. Therefore, this study has established sexual and asexual S. mediterranea as simple, yet attractive models for investigating energetic costs between sexual and asexual phenotypes. This research also provided quantitative values for specific dynamic action in planaria, with a maximum increase in oxygen consumption of 160% induced by feeding, as well as metabolic relationships in planaria involving temperature, age, and regeneration. These values establish planaria as one of the simplest animal models in which common metabolic patterns, such as SDA and poikilothermic temperature sensitivity, have been demonstrated. Therefore, this research has contributed to the overall knowledge of the basic physiology in this animal, providing the framework for future metabolic studies in planaria involving environmental factors, reproduction, regeneration, development, and aging. Information from this study may supplement interpretation and understanding of modern cellular, molecular, and genomic studies in planaria.

Hepcidin, the critical iron regulatory factor, is a small peptide produced by the hepatocytes in response to increased body iron and inflammation. Circulating hepcidin controls both intestinal iron absorption and the release of iron from macrophages into plasma via a negative iron feedback system. I developed a novel competitive immunoassay for hepcidin using a polyclonal antibody produced against synthetic hepcidin. I validated the immunoassay and determined it was able to discriminate between healthy controls and selected disease groups. I compared the immunoassay against another established method of measuring hepcidin. I established that plasma hepcidin has a diurnal rhythm and that plasma hepcidin increases in response to intravenous iron in anaemic patients. Elevated levels of hepcidin in renal failure may have a role in the erythropoietin resistance observed in renal anaemia. In haemodialysis patients, hepcidin levels were significantly elevated, but there was no correlation with inflammatory markers. Elevated hepcidin was associated with anemia, but erythropoietin dose was negatively correlated with hepcidin, suggesting that erythropoietin suppresses hepcidin levels. This was confirmed in patients when hepcidin levels significantly decreased after erythropoietin treatment. The association between plasma hepcidin and other iron parameters were also examined in healthy controls after erythropoietin administration and venesection. Profound hepcidin suppression was observed after an erythropoietin dose, with peak levels reduced by 73.2%, and then gradually recovering over the following two weeks. A similar but more gradual change in hepcidin was observed after reducing hematocrit by removal of 250 mL blood. The studies suggested that the marrow–hepcidin axis is regulated by factors other than those specifically investigated. In summary, I have developed and validated a novel immunoassay for hepcidin which will allow further investigation of the vital role of this peptide in iron homeostasis and human physiology.

The use of islets isolated from the human fetal pancreas may provide a source of transplantable tissue with the potential to reverse diabetes. Further research is required before there is enough knowledge of the function of these islets to enable their successful transplantation to humans. Chapter 1 Experiments on fetal pancreas are limited by the paucity and irregularity of the supply of donor tissue. Therefore, a human fetal pancreatic B—cell line would be of considerable use in the better understanding of this potential source of transplantable tissue. In chapter 1 of this thesis, success in isolating an immortalised fetal lung fibroblast cell line by SV—40 viral infection is described. Satisfied with this procedure, attempts were then made to isolate a human fetal pancreatic .cell line by infection with SV—4O virus and transfection with plasmid vectors containing SV—4O genes. Although unsuccessful in isolating an immortalised cell line, a protocol was established for the transfection of human fetal endocrine cells in the future. Chapter 2 It has been well established that the fetal rat and human pancreatic islet B—cell secretes insulin poorly in response to glucose compared to that from adult B—cells. However, the cellular mechanism for this is unknown. In chapter 2, microfluorometric studies are described using the intracellular fluorescent dye fura—Z to measure changes in the cytoplasmic free Ca2+ concentration in fetal, neonatal and adult rat B—cells to determine the ability of glucose and other insulin secretagogues to cause an increase in [Ca2+]i believed to be the trigger for insulin secretion. The fetal B-cell responded to various insulin secretagogues, but not glucose, with an increase in [Ca2+].l and insulin release. The immature insulin secretory response to glucose by the fetal B—cell is, therefore, due to its inability to translate glucose stimulation into an increase in [Ca2+]i required for exocytosis of insulin. Chapter 3 The protocols used in experiments conducted on rat fetal B-cells were used for experiments on human fetal B—cells described in chapter 3 to determine the ability of glucose and other insulin secretogogues to cause an increase in [Ca“1i. In this study, mid-gestation fetal human and late—gestation fetal porcine islet—like cell clusters, as well as adult human islets and the only available human B—cell line, the adult insulinoma cell line HP—62, were studied using microfluorometric methods. Both the mid—gestation human fetal B-cell and the late—gestation porcine fetal B—cell possesses the complete mechanism required to translate stimulation by secretagogues other than glucose into an increase in [Ca2+]i required for secretion of insulin. The end-stage of the signal transduction pathway in these fetal B—cell is, therefore, mature. The immature secretory response to glucose must, therefore, be located at some other step of glucose-induced stimulation such as glucose transport, glucose metabolism or in the linkage between glycolysis and mitochondrial metabolism. Chapter 4 The amino acid arginine is known to cause insulin release from glucose—insensitive human and rat fetal B—cells, as well as from rat adult perfused pancreas, pancreatic explants and perifused islets. Exposure of rat and human fetal B—cells to arginine was found in the studies described in chapter 2 and 3 to cause an increase in [Ca“]r The cellular mechanisms involved in the B—cell response to arginine are not well described. Experiments described in chapter 4 were conducted to identify the mechanisms involved in arginine-induced stimulation of the B—cell using an insulinoma cell line (NIT—l). As in the studies described. in Chapters 2 and 3, the ratiometric fluorescent probe of Ca”) fura—Z, was used to directly monitor changes in [Ca”1i. The effects of L—arginine, and its analogues which do not produce nitric oxide were investigated to characterise the mechanisms of arginine—induced B—cell stimulation and determine if nitric oxide production plays a role in this stimulation. The data demonstrate that L—arginine, and its analogues, caused similar increases in [Ca2+]i and insulin release. This suggests that nitric oxide production has no role in the arginine—induced increase in [Ca2+]i in the NIT—l cell. The increase in [Ca2+]i is due to a combination of the direct depolarisation of the membrane due to the electrogenic effect of arginine plus an effect due to the metabolism of the amino acid.

Students Learn when they are actively engaged and thinking in class. The activities in this book are the primary classroom materials for teaching Anatomy and Physiology, sing the POGIL method. The result is an "I can do this" attitude, increased retention, and a feeling of ownership over the material.
https://dc.etsu.edu/etsu_books/1027/thumbnail.jpg

Organ Systems & Translational Medicine
Ph.D.
Platelets are involved in many processes ranging from fighting microbial infections and triggering inflammation to promoting tumor angiogenesis and metastasis. Nevertheless, the primary physiological function of platelets is to act as essential mediators in maintaining homeostasis of the circulatory system by forming hemostatic thrombi that prevent blood loss and maintain vascular integrity. The p21-activated kinases (PAKs) are a family of serine/threonine kinases known to be the downstream effectors of GTPases, Cdc42 and Rac1. PAKs are the key regulators of actin polymerization and have been shown to play an important role in platelet spreading and aggregation in thrombin-stimulated platelets. Whereas several signaling cascades downstream of heterotrimeric G proteins that regulate platelet functions have been characterized, little attention is paid towards the signaling cascades that involve small G proteins effectors such as PAK. A few studies have characterized the role of PAK, downstream of the Rho family of small G proteins, in outside-in signaling, but its role in the regulation of platelet functional responses by inside-out signaling events have not been elucidated. PAK is reported to interact with numerous proteins including Akt, PDK1 and PI3-kinase in different cell lines. PAK’s function as a scaffolding protein expands the role of this protein in cellular functions. Although PAK is known to have non-catalytic scaffolding functions and is shown to associate and translocate Akt in other cell systems, the catalytic and possible non-catalytic scaffolding role in platelet functions are not clearly defined. In this dissertation we propose to elucidate the scaffolding function of PAK and also its role platelet functional responses using molecular genetics approach. Akt is an important signaling molecule regulating platelet aggregation. Akt is phosphorylated upon translocation to the membrane through Gi signaling pathways by a PIP3-dependent mechanism. However, Akt is more robustly phosphorylated by thrombin compared to ADP in platelets. In this study, we investigated the mechanisms of Akt translocation as a possible explanation for this difference. Stimulation of washed human platelets with protease-activated receptor (PAR) agonists caused rapid translocation of Akt to the membrane, whereas Akt phosphorylation occurred later. The translocation of Akt was abolished in the presence of a Gq-selective inhibitor or in Gq-deficient murine platelets, indicating that Akt translocation is regulated downstream of Gq signaling pathways. Interestingly, PI3-kinase inhibitors or P2Y12 antagonist abolished Akt phosphorylation without affecting Akt translocation to the membrane, suggesting that Akt translocation occurs through a PI3-kinase/PIP3/ Gi-independent mechanism. An Akt scaffolding protein, PAK, translocates to the membrane upon stimulation with PAR agonists in a Gq-dependent manner with the kinetics of translocation similar to that of Akt. Co-immunoprecipitation studies showed constitutive association of PAK and Akt, suggesting a role of PAK in Akt translocation. These results show for the first time an important role of the Gq signaling pathway in mediating Akt translocation to the membrane in a novel Gi/PI3-kinase/PIP3-independent mechanism. PAK contains an autoinhibitory domain that suppresses the catalytic activity of its kinase domain. This autoregulatory domain found within PAK kinase provides a unique target for chemical inhibitors. IPA3, a small molecule allosteric inhibitor of PAK activation, binds covalently to the PAK regulatory domain and prevents binding to its upstream activators. IPA3 has been used in various cells, including platelets, to evaluate the role of PAK in signaling. Herein, we investigated the specificity and selectivity of IPA3 as a PAK inhibitor in the human platelets. Stimulation of platelets pretreated with IPA3 using a PAR-4 or GPV1 agonist resulted in a concentration dependent inhibition of aggregation, as was suggested by earlier studies. Interestingly, we found that incubation of washed human platelets with IPA3 leads to a non-specific increase in phosphorylation of several proteins in absence of any agonist. However, this phosphorylation is not sufficient for aggregation of platelets by IPA3. In summary, we demonstrate that IPA3 by itself can phosphorylate several proteins in human platelets and thus its use is not an appropriate strategy for investigating PAK function in platelets. PAKs are classified into two groups based on their structural differences. Human platelets have been shown to express both group I (PAK1, PAK2, and PAK3) and group II PAKs (PAK4). Previous studies showing the role of PAK were performed with nonspecific inhibitors of PAK, such as IPA3, that do not distinguish isoforms. Thus, we propose to evaluate the function of specific PAK isoforms in platelets using knockout murine platelets, which are more selective tools to study the role of individual isoforms of PAK. We observed that Pak2 null mice showed enhanced secretion responses upon stimulation with 2MeS ADP and collagen, and delayed clot retraction. Interestingly, Pak1 null murine platelets did not have any functional defects, suggesting redundancy with other PAK isoforms. The studies proposed in my thesis will provide further insights into the molecular mechanisms of platelet activation and hence provide a basis for development of PAK as novel antithrombotic therapeutic targets. Furthermore, PAK inhibitors are currently being developed by pharmaceutical companies to treat malignancies, although this enzyme is ubiquitously expressed in the body. A thorough understanding of the role of PAK in platelets can predict the effect of these drugs on hemostatic functions, which helps during clinical trials. In the future, targeted inhibition of signaling molecules in platelets could be developed and that would solely target platelet signaling pathways.
Temple University--Theses

The research in the field of industrial biotechnology, especially regards bioproducts and bioprocesses, are aimed at developing innovative technologies that lead to obtaining compounds with the use of microorganisms, or seeking to enhance existing processes to increase yield, production and productivity, trying to ensure a higher degree of sustainability and reducing environmental impact. To pursue these goals is possible to intervene by adopting a “technologic” approach that includes the development of systems capable of ensuring a more effective control of the parameters that govern the production processes or, with a “molecular / metabolic” approach, acting directly on the host system, namely by working on production capacity of the microorganism itself. To achieve this goal the pathways responsible for the processes of synthesis or secretion of products of interest must be identified and, if necessary, modified, or the environmental conditions in which the organism is operating during the process must be considered, to study how to improve its production capacity even in non-optimal physiological condition. The process conditions that characterize industrial production processes often put the cells through a series of stress that inevitably act negatively on yields. It therefore becomes necessary to identify the limiting factors in relation to the host organism and, on this basis, to act in an appropriate manner. To do this it is possible to adopt different strategies, complementary and not mutually exclusive. The most immediate is the exploration of biodiversity in order to choose a host that is intrinsically and naturally more resistant to the type of stress imposed by the process. This way is not always easy to follow, given the extent of the possible solutions and the lack of resources that allow the exploration and characterization in a reasonable time for the development of a biotechnological process. An alternative strategy is focused on the characterization of the cellular response to these stress conditions to identify the key factors involved in the mechanism. In this way, for example, through genetic manipulation on these factors it could be possible to improve the resistance of the cell itself, or by the transfer of these specific genetic traits to improve the resistance of other micro-organisms selected as host system. The yeast Saccharomyces cerevisiae is one of the most widely used microorganisms for the production of compounds of biotechnological interest because of the available knowledge on the physiology, genetics, biochemistry, and on the existence of technological and molecular tools suitable for its manipulation in order to optimize the production by fermentation. It is important to underline that S. cerevisiae is recognized as a GRAS organism (generally regarded as safe) by the Food & Drug Administration that has allowed the use for the production of pharmaceutical compounds for human use. While it is now increasingly clear the potential of S. cerevisiae as a platform for metabolic engineering, for some heterologous proteins production on a large scale this yeast is not the ideal host system. Very often the expressed proteins are hyper-glycosilated or, if retained in the periplasmic space, they suffer significant degradation. For other industrial productions, especially where the product of interest has to be cheap, the fermentation technologies needed are too complex and sophisticated (and expensive) to be implemented on a large scale. With these assumptions in recent years it has been explored the opportunity of adopting other yeasts, called "unconventional yeast" by developing new systems of expression. In this thesis project an "alternative" yeast, Zygosaccharomyces bailii, is considered. Although it is not well characterized from molecular and genetic point of view, it presents interesting features in view of potential biotechnological production: it allows high yields of biomass, it has a high specific growth rate and a higher resistance compared to S. cerevisiae, to certain types of stress, and in particular to stresses generated by an acid medium. The cell surface, as an area of communication and exchange between the extracellular environment and the cell itself is one of the targets of this study. In S. cerevisiae, thanks to the availability of molecular tools and the knowledge of the entire genome sequence, it is possible the design of in-depth studies and the engineering of the cellular metabolic network. Given the difficulties in its genetic manipulation, in the case of Z. bailii was first necessary to address another preliminary issue. In this diploid yeast the deletion of an essential gene was never been made. It has been developed a reproducible protocol for gene deletion by a gene-targeting approach and it was obtained the deletion of the gene ZbLEU2, who gave birth to the first auxotroph strain of Z. bailii (leuˉ). This represents an important step for a possible use of Z. bailii as host system. Thanks to this protocol a mutant strain of Z. bailii of potential interest for heterologous protein production was also obtained, in analogy with what reported in the literature for S. cerevisiae. The deletion concerns the homologue of ScGAS1, coding for the enzyme β-1 ,3-glucanosyltransferase which catalyzes the crosslinking of cell wall glucans. ZbGAS1. The gene was cloned by PCR and sequenced. The deleted mutant of Z. bailii has morphological and phenotypic characteristics very similar to the correspondent in S. cerevisiae, showing an alteration of the cell wall structure, and enhanced secretive capacity than the wild type strain for some heterologous proteins that have been considered. In parallel to these studies, populations of Z. bailii growing on different carbon sources were analyzed by flow cytometry. The analysis of DNA and protein content was performed to better characterize this yeast not only from molecular point of view, but also to explore its cellular features. The characterization of this unconventional yeast confirmed once again one of the most appreciated features for yeasts used as cell factories: versatility. This property is so strong that yeasts has been exploited for natural abilities, such as production of ethanol, and also for processes where a targeted manipulation was introduced, for example in lactic acid production, just to cite a pair of biotechnological production of industrial relevance. To make this process competitive on the market, in terms of yield, production and productivity, yeasts were pushed to their physiological limits. These limits are given by the accumulation of vast amounts of product that, in the case of ethanol, can cause damage to the lipid component of the plasma membrane, in the case of lactic acid can result in a loss of proper cellular homeostasis with the fall of the intracellular pH. It is therefore necessary to assess whether these limitations can be overcome by acting in particular on the plasma membrane, whose role in controlling the transport and the cellular homeostasis makes it a target of interest to improve the robustness of cells in response to stress generated by the process, such as oxidative stress or generated by low pH. The optimization of transport through the membrane plays a key role in the mechanisms of adaptation to these stresses. In particular, improving the flux of nutrients entering the cell could allow an optimal uptake of nutrient in the cytoplasm (as in the case of bioethanol production), an improvement of outflows from the cells might instead allow effective removal of compounds that may be deleterious for cell viability when present beyond a threshold value (eg organic acids). By focusing on the protein fraction that characterizes the cytoplasmic membrane, we have studied the effects of modulation of the expression of H +-ATPase pump of the plasma membrane (Pma1p), involved in the intracellular pH homeostasis. In particular, the gene ScPMA1 was overexpressed in S. cerevisiae and this overexpression was able to confer a greater resistance to acid stress, evidenced by growth kinetics in the presence of lactic acid. The increased cell viability under restrictive conditions in respect to the wild type strain was also checked by flow cytometry. The use of this tool has enabled the development of a system able to assess quantitatively the degree of robustness of the cells in stressful conditions. With this instrument able to assess the robustness of the cells as a function of various types of stress (oxidative, pH, ...) it is possible to design new interventions of metabolic engineering in order to provide greater resistance to yeast in restrictive process conditions, similar to conditions prevailing in the processes of production of compounds of biotech interest. It will be possible to evaluate the effectiveness of these interventions with the flow cytometer by assessing the response of the engineered cells under restrictive conditions by measuring the ability of cells to increase their robustness. The robustness remains one of the key features of yeasts as microbial cell factories, in particular to reach one of the main goal of White Biotechnology: to provide value products from renewable resources through sustainable processes with low environmental impact.